Methods of using nonpolar grape seed extracts to stimulate tyrosinase expression or melanin synthesis

ABSTRACT

The present invention relates to methods of increasing tyrosinase synthesis, expression, or activity in a cell comprising administering to the cell at least one non polar grape seed extract, for example, a nonpolar grape seed extract obtained using hexane as an extraction solvent. The nonpolar grape seed extract may be obtained by any extraction process including solvent sequential fractionation and supercritical fluid extraction.

The present application is a continuation-in-part and claims priority toU.S. patent application Ser. No. 11/300,095, filed Dec. 14, 2005, whichclaims priority to U.S. Provisional Application Ser. No. 60/635,763,filed Dec. 14, 2004 and to PCT Application No. PCT/US2005/045149 filedDec. 14, 2005.

BACKGROUND

The present invention relates to methods of stimulating tyrosinaseexpression or melanin synthesis in the skin or hair of a mammalcomprising topically applying to the skin or hair of the mammal acomposition comprising at least one nonpolar grape seed extract(hereafter “nonpolar GSE”), for example, a nonpolar GSE obtained usinghexane as an extraction solvent by itself, or as an extraction solventafter extraction with acetone (hereafter “hexane subfraction”), and acosmetically suitable vehicle.

A lack of gray or graying hair is generally associated with a youthfulor aesthetically pleasing appearance. A sun tanned skin is alsogenerally associated with a youthful or aesthetically pleasingappearance. However, there are disadvantages associated with currentmethods for covering gray or graying hair and for increasing a tannedappearance.

For example, typically, one will use a temporary or permanent hair dyeto mask or cover the gray hair. These dyes, however, can be harsh, dryor damage hair, and typically fade and grow out over time, leading todull hair and gray hair roots. Similarly, a tan appearance is typicallyachieved by either repeated or prolonged exposure of the skin to—ultraviolet rays in sunlight or to chemical and natural stains. Inrecent years, prolonged exposure to ultra-violet rays, both natural andartificial, has been associated with life-threatening disorders such asmelanoma or various other forms of skin cancer. Prolonged exposure toultra-violet rays is also associated with undesirable characteristicssuch as wrinkled or prematurely aged skin. Chemical and natural stainsfor the skin may result in a non-natural skin color, may have anunpleasant odor, usually only provide a short duration of tanappearance, and are generally difficult to apply.

BRIEF SUMMARY

The various hues and degrees of pigmentation found in the skin and hairof mammals are directly related to the amount of melanin present in theskin or hair. Melanin is a pigment produced by melanocytes, which arecells found among the basal cells of the epidermis. The synthesis ofmelanin is catalyzed by the enzyme tyrosinase, which is expressedpreferentially in melanocytes. Higher levels of tyrosinase geneexpression correlate to higher levels of melanin. Higher levels ofmelanin in the skin and hair of a mammal correlate to darker hair andskin color.

It has been discovered that a nonpolar GSE, for example a nonpolar GSEobtained using hexane as an extraction solvent or subfraction solvent,directly stimulates tyrosinase gene expression and results in melaninsynthesis in melanocytes when topically applied. Accordingly, in oneembodiment, the present invention provides a method for stimulatingtyrosinase gene expression or melanin synthesis in the skin of a mammalthat comprises topically applying a composition comprising at least onenonpolar GSE, for example a nonpolar GSE obtained using hexane as anextraction solvent or subfraction solvent, and a cosmetically suitablevehicle, to produce an increased tan appearance.

In another embodiment, the invention provides a method that includestopically applying a composition of a nonpolar GSE, for example anonpolar GSE obtained using hexane as an extraction solvent orsubfraction solvent, and a cosmetically suitable vehicle to the scalp ofa mammal or other location where gray hair is present, to increasetyrosinase gene expression or melanin synthesis and thereby decreasegray appearance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart illustrating the regulation of the tyrosinase genemediated by GSE in G-361 melanocyte-like cells.

FIG. 2 is a chart illustrating the effect of various GSE fractions ontyrosinase gene expression in the G-361 melanocyte-like cell line. InFIG. 2 “1-EtOH” represents an extract of a grape seed obtained usingethanol as the solvent in a total extraction procedure; “2-EtOH.H20”represents an extract of a grape seed obtained using ethanol/water assolvents in a total extraction procedure; “Hexane” represents an extractof a grape seed obtained using hexane as a solvent in a solventsequential fractionation procedure; “Ethyl acetate” represents anextract of a grape seed obtained using ethyl acetate as a solvent in asolvent sequential fractionation procedure; “3C-Ethanol” represents anextract of a grape seed obtained using ethanol as a solvent in a solventsequential fractionation procedure; “3D-EtOH.H20” represents an extractof a grape seed obtained using ethanol/water as solvents in a solventsequential fractionation procedure; “Chloroform” represents an extractof a grape seed obtained using chloroform as an extract; “Acetone”represents an extract of a grape seed obtained using acetone as anextract; and “Grav” represents a control GSE that was not subjected toany further extraction or fractionation procedures.

FIG. 3 is a chart illustrating the effect of various GSE fractions,including a GSE fraction obtained using acetone as an extractionsolvent, and GSE subfractions obtained using methanol and hexane asextraction solvents, on tyrosinase gene expression in the G-361melanocyte-like cell line.

FIG. 4 is a chart illustrating tyrosinase inhibition activity forvarious GSE fractions. The data represent inhibition activity per gramof each fraction. Kojic Acid is presented as a reference, usinginhibited units per milligram to accommodate the scale. In FIG. 4“EtOH-1” represents an extract of a grape seed obtained using ethanol asthe solvent in a total extraction procedure; “EtOH/H20-2” represents anextract of a grape seed obtained using ethanol/water as solvents in atotal extraction procedure; “Hex-3A” represents an extract of a grapeseed obtained using hexane as a solvent in a solvent sequentialfractionation procedure; “EtAc-3B” represents an extract of a grape seedobtained using ethyl acetate as a solvent in a solvent sequentialfractionation procedure; “EtOH-3C” represents an extract of a grape seedobtained using ethanol as a solvent in a solvent sequentialfractionation procedure; “EtOH/H20-3D” represents an extract of a grapeseed obtained using ethanol/water as solvents in a solvent sequentialfractionation procedure; “CHCI3-4” represents an extract of a grape seedobtained using chloroform as an extract; “Ace-5” represents an extractof a grape seed obtained using acetone as an extract; “SFE” representsan extract of a grape seed obtained using supercritical fluidextraction; “blank” represents a control; and “buffer” representsanother control.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to theparticular methodology or protocols described herein. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by the claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

The following definitions are provided in order to aid the reader inunderstanding the detailed description of the present invention, as wellas to aid in the understanding of the appended claims.

As used herein, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to “a nonpolar GSE” is a reference to one or morenonpolar GSEs and includes equivalents thereof known to those skilled inthe art, and so forth.

“Nonpolar GSE” and the phrase “nonpolar grape seed extract” refer to anynonpolar compound or combination of nonpolar compounds obtained fromgrape seed and/or to any nonpolar oils obtained from grape seed, as wellas to chemical derivatives thereof. In one example, a nonpolar GSE isobtained using hexane as an extraction solvent.

There are numerous extraction methods that can be used to produce anonpolar GSE suitable for use in the present invention. These extractionmethods are described herein, well-known in the art, and may bedescribed in various publications and patents. For example, solventsequential fractionation with a series of organic solvents may be usedto obtain a nonpolar GSE. In one example, a nonpolar GSE is obtained byfirst using acetone as an extraction solvent and then subsequently usinghexane as an extraction solvent. Analytical studies such asUltraviolet-Visible Spectroscopy and High Performance LiquidChromatography have concluded that there are no polar compounds such asmonomeric phenols and oligomeric proanthocyanidins found in asubfraction obtained by first using acetone as an extraction solvent andsubsequently using hexane as an extraction solvent.

“Proanthocyanidins” are polar flavonoids that are commonly found in mostcommercially available grape seed extracts. Proanthocyanidins havewell-known antioxidant properties and are procyanidolic oligomers.Typically, proanthocyanidins are known for their ability to inhibittyrosinase. For example, see U.S. Pat. No. 6,590,105 and JP 2000-159681by Hai Tai Confectionary Co.

“Tyrosinase” is an enzyme that plays a role in the melanin synthesispathway and therefore its activity directly impacts the amount ofmelanin produced. Tyrosinase is expressed in specialized cells calledmelanocytes. Melanocytes encapsulate the active enzyme in subcellularorganelles called melanosomes. Mature melanosomes are transferred tokeratinocytes and melanin is produced. An inherited lack of tyrosinaseactivity results in one of the forms of albinism.

“Melanin” is a water-insoluble polymer derived from the amino acidtyrosine. It is one of two pigments found in mammalian skin and hair andadds brown/black, and red/yellow to skin and hair color pigmentsnaturally produced by melanocytes. The degree of pigmentation isdependent on both the amount and type of melanin produced and on thephysical distribution of melanosomes.

The present invention is based on the surprising discovery that topicalapplication of at least one nonpolar GSE, for example, a nonpolar GSEobtained using hexane as an extraction solvent, and a cosmeticallysuitable vehicle directly stimulates tyrosinase gene expression andresults in increased melanin synthesis. Prior to this discovery, it wasgenerally understood that polar compounds, such as catechins andproanthocyanidins, present in grape seed extracts are typically knownfor their ability to exert an effect on tyrosinase and melaninsynthesis. See e.g. JP 200-159,105 and JP 2000-159681; Shi et al.,“Polyphenolics in grape seeds-biochemistry and functionality.” Med Food.2003 Winter;6(4):291-9.

The current state of the art concerning nonpolar GSE does not recognizethe unique ability of nonpolar GSE to directly stimulate tyrosinaseexpression and thereby lead to an increased tan appearance and adecreased appearance of gray hair. Thus, the present invention providesa method that includes topically applying a composition comprising atleast one nonpolar GSE, for example, a nonpolar GSE obtained usinghexane as an extraction solvent or subfraction solvent, and acosmetically suitable vehicle to the skin or hair of a mammal tostimulate tyrosinase expression in the vicinity of the topicalapplication, resulting in an increased tan appearance or in a decreasedappearance of gray hair.

In one embodiment, the present invention includes administration of atleast one nonpolar GSE at a dose ranging from approximately 30 toapproximately 150 μg/ml. In another embodiment, the present inventionincludes administration of at least one nonpolar GSE at a dose rangingfrom approximately 45 to approximately 75 μg/ml. In a furtherembodiment, the present invention includes administration of at leastone nonpolar GSE at a dose of approximately 50 μg/ml.

To prepare the compositions according to the present invention, at leastone nonpolar GSE, for example, a nonpolar GSE obtained using hexane asan extraction solvent or subfraction solvent, may be mixed with acosmetically suitable vehicle. The nonpolar GSE may be present in anamount from about 0.01% to about 10% by weight of the total composition.At least one nonpolar GSE may also be present in an amount from about 1%to about 9% by weight of the total composition. Alternatively, at leastone nonpolar GSE may be present in an amount from about 2% to about 8%by weight of the total composition. At least one nonpolar GSE may alsobe present in an amount from about 3% to about 7% by weight of the totalcomposition. In another alternative embodiment, at least one nonpolarGSE is present in an amount from about 4% to about 6% by weight of thetotal composition. In one example of a preferred embodiment of thepresent invention, at least one nonpolar GSE is present in an amountfrom about 2% to about 3% by weight of the total composition.

Additionally, it is preferable that the cosmetically suitable vehicleforms from about 90% to about 99.99% by weight of the total composition,and more preferably from about 97% to 99% by weight of the totalcomposition. The cosmetically suitable vehicle may also form from about91% to about 98% by weight of the total composition; from about 92% toabout 97% by weight of the total composition; from about 93% to about96% by weight of the total composition; or from about 94% to about 95%by weight of the total composition. The cosmetically suitable vehiclecan, in the absence of other cosmetic adjuncts or additives, form thebalance of the composition.

The composition of the present invention may be formulated as asolution, gel, lotion, cream, ointment, oil-in-water emulsion,water-in-oil emulsion, stick, spray, paste, mousse, tonic, or othercosmetically suitable form.

Extracts of grape seeds from grapes of any color or variety may be used.One of ordinary skill in the art will appreciate that generally, redgrape seeds have a higher total polyphenolic content than white grapeseeds; 3500 mg/kg on average for reds compared to 2800 mg/kg on averagefor whites. See Grape Seed Extract White Paper. Prepared by: The GrapeSeed Method Evaluation Commiftee (available at 222.activin.com/Testing%20White %20pater_.html). In one example, grape seeds used in obtainingthe nonpolar GSE to be used in the present invention can be acquiredfrom either grape juice operations or from wine producers after theyhave been discarded from the winemaking process.

Additionally, the nonpolar GSE used in practicing the present inventionmay be obtained from any commercially available source.

One of ordinary skill in the art will also appreciate that the nonpolarGSE used in practicing the method of the present invention can be madeusing several extraction methods. For example, a nonpolar GSE can beproduced by extracting grape seed with an organic solvent. Hexane is anexample of an organic solvent that might be used in producing the GSE tobe used in the present invention.

In another example, solvent sequential fractionation may be used toextract nonpolar GSE. For example, using this technique, grape seeds canbe sequentially extracted with acetone,and hexane. The extracts obtainedafter each step (fractions) of the sequence will contain chemicalcompounds in decreasing order of polarity similar to the solvents usedfor extracting them. The fractions are dried to evaporate the solvents,resulting in a nonpolar GSE. Those of skill in the art will appreciatethat many other solvents can be used in practicing the solventsequential fractionation extraction of a nonpolar GSE.

Another example of an extraction technique that might be used to obtainGSE is supercritical fluid carbon dioxide extraction (SFE). In thisextraction procedure the grape material is not exposed to any organicsolvents. Rather, the extraction solvent is carbon dioxide, with orwithout a modifier, in supercritical conditions (>31.3° C. and >73.8bar). Those of skill in the art will appreciate that temperature andpressure conditions can be varied to obtain the best yield of extract.This technique generates a GSE of fat soluble and/or lipophiliccompounds, similar to the total hexane extraction technique describedabove.

Another extraction technique that may be used to obtain a nonpolar grapeseed extract is solid phase extraction (SPE) or reverse-phase SPE.Typically this technique involves use of a solid sorbent material,typically an alkyl bonded silica, which is packed into a cartridge orimbedded in a disk. This solid sorbent material performs essentially thesame function as an organic solvent in liquid-liquid extraction. Forexample, reverse-phase SPE may be employed to extract non-polarcompounds from polar samples such as water by utilizing a solid sorbentcontaining non-polar functional groups such as octadecyl (C18) or octyl(C8) bonded silicas. Aqueous samples are pumped or pulled through thecartridge or disk and organic compounds in the samples interact with thenon-polar functional groups on the sorbent and are effectively extractedfrom the sample. Organic compounds in the original aqueous sample may beeluted from the cartridge or disk with a small volume of organicsolvent.

Any of the above extraction techniques, or other extraction processesand techniques in the art, may be used as part of a bio-directedfractionation process. Bio-directed fractionation focuses on determiningwhich fraction of a GSE is responsible for the various known GSEactivities. Therefore, in one example, a bio-directed fractionationtechnique may be used to separate polar and non-polar constituents of aGSE. In another example, GSE may be used to separate GSE constituentsthat increase tyrosinase synthesis and/or expression from GSEconstituents that inhibit tyrosinase synthesis and/or expression.

Those of skill in the art will appreciate that there are many otherextraction and fractionation processes, both known in the art anddescribed in various patents and publications that can be used to obtainthe nonpolar GSE to be used in practicing the present invention. Forexample, the extraction procedures described in the followingreferences, which are incorporated herein by reference, could be used inpracticing the present invention: Murga et al., “Extraction of naturalcomplex phenols and tannins from grape seeds by using supercriticalmixtures of carbon dioxide and alcohol.” J. Agric Food Chem. 2000Aug:48(8):3408-12; Hong et al., “Microwave-assisted extraction ofphenolic compounds from grape seed.” Nat Prod Lett. 2001; 15(3):197-204; Ashraf-Khorassani et al., “Sequential fractionation of grapeseeds into oils, polyphenols, and procyanidins via a single systememploying CO₂-based fluids.” J. Agric Food Chem., 2004 May5;52(9):2440-4.

The nonpolar GSE to be used in practicing the present invention may bedelivered topically by any means known to those of skill in the art. Forexample, the nonpolar GSE might be delivered using a cosmeticallysuitable vehicle. A cosmetically suitable vehicle may act variously assolvent, carrier, diluent or dispersant for the constituents of thecomposition, and allows for the uniform application of the constituentsto the surface of a cell or the skin at an appropriate dilution. Thecosmetically suitable vehicle may also facilitate penetration of thecomposition into the skin, scalp, or hair follicle.

The nonpolar GSE used in practicing the present invention may be solubleor insoluble in the cosmetically suitable vehicle. If the at least onenonpolar GSE is soluble in the cosmetically suitable vehicle, then thevehicle acts as solvent for the nonpolar GSE. However, if the at leastone nonpolar GSE is insoluble in the cosmetically suitable vehicle, thenit is dispersed in the vehicle by means of, for example, a suspension,emulsion, gel, cream or paste, and the like.

Thus, it will be apparent to the skilled artisan that the range ofpossible cosmetically suitable vehicles is very broad. For example,cosmetically suitable vehicles can be emulsions, lotions, creams, ortonics. Cosmetically suitable vehicles can comprise water, ethanol,butylene glycol, or other various solvents that aid in penetration ofthe skin or hair follicle. Some examples of suitable vehicles aredescribed in U.S. Pat. No. 6,184,247 and in U.S. Pat. No. 6,579,516 theentire contents of which are incorporated herein by reference.

Preferably the cosmetically suitable vehicle used in practicing thepresent invention comprises water and ethanol. Optionally, thecosmetically suitable vehicle also contains butylene glycol and/orfrescolate MGA. For example, the cosmetically suitable vehicle cancomprise approximately 40-60% water, approximately 45-55% ethanol, andapproximately 5-10% % butylene glycol by weight of the totalcomposition. In practicing the present invention, preferably thiscosmetically suitable vehicle is mixed with at least one nonpolar GSEcomprising approximately 2% by weight of the total composition. In otherembodiments, the cosmetically suitable vehicle is mixed with at leastone nonpolar GSE comprising approximately 0.01% to 10% by weight of thetotal composition; approximately 0.05% to 9% by weight of the totalcomposition; approximately 1% to 8% by weight of the total composition;approximately 2% to 7% by weight of the total composition; orapproximately 3% to 6% by weight of the total composition.

In general, however, cosmetically suitable vehicles according to thepresent invention may comprise, but are not limited to comprising, anyof the following examples: water; castor oil; ethylene glycol monobutylether; diethylene glycol monoethyl ether; corn oil; dimethyl sulfoxide;ethylene glycol; isopropanol; soybean oil; glycerin; soluble collagen;zinc oxide; titanium oxide; Kaolin; or hyaluronic acid.

Additionally, cosmetically suitable vehicles used in the presentinvention may optionally comprise one or more humectants, including butnot limited to: dibutyl phthalate; gelatin; glycerin; soluble collagen;sorbitol; or sodium 2-pyrrolidone-5-carboxylate. Other examples ofhumectants that may be used in practicing the present invention arefound at page 575 of the CFTA Cosmetic Ingredient Dictionary andHandbook (10^(th) ed.), and are incorporated herein by reference.

Additionally, cosmetically suitable vehicles in the present inventionmay optionally comprise one or more emollients including but not limitedto: butane-1,3-diol; cetyl palmitate; dimethylpolysiloxane; glycerylmonoricinoleate; glyceryl monostearate; isobutyl palmitate; isocetylstearate; isopropyl palmitate; isopropyl stearate; butyl stearate;isopropyl laurate; hexyl laurate; decyl oleate; isopropyl myristate;lauryl lactate; octadecan-2-ol; caprylic triglyceride; caprictriglyceride; polyethylene glycol; propane-1,2-diol; triethylene glycol;sesame oil; coconut oil; safflower oil; isoamyl laurate; nonoxynol-9;panthenol; hydrogenated vegetable oil; tocopheryl acetate; tocopheryllinoleate; allantoin; propylene glycol; arachis oil; castor oil;isostearic acid; palmitic acid; isopropyl linoleate; lauryl lactate;myristyl lactate; decyl oleate; or myristyl myristate. Other examples ofemollients that may be used in practicing the present invention arefound at pages 572-575 of the CFTA Cosmetic Ingredient Dictionary andHandbook (10^(th) ed.), and are incorporated herein by reference.

Additionally, cosmetically suitable vehicles used in the presentinvention may optionally comprise one or more penetration enhancersincluding but not limited to: surfactants such as pyrrolidones andalkanols., for example, 2-pyrrolidone or decanol; alcohols, such asethanol; glycols, such as propylene glycol, dipropylene glycol, butyleneglycol, ethoxydiglycol; glycerin; and terpenes.

Other cosmetically suitable vehicles that may be used in practicing thepresent invention will be apparent to those of skill in the art and areincluded within the scope of the present invention.

For example, a cosmetically suitable vehicle can be a lotion that istopically applied. In one example, the lotion may comprise cabomer 981,water, glycerin, isopropyl myristate, mineral oil, shea butter, stearicacid, glycol stearate, cetyl alcohol, dimethicone, preservatives, tea,and nonpolar GSE.

The nonpolar GSE compositions of the present invention may also containvarious known and conventional cosmetic adjuvants so long as they do notdetrimentally affect the desired melanin or tyrosinase stimulatingeffect provided by the at least one nonpolar GSE. For example, thecomposition of the present invention can further include one or moreadditives or other optional ingredients well known in the art, which caninclude but are not limited to fillers (e.g., solid, semi-solid, liquid,etc.); carriers; diluents; thickening agents; gelling agents; vitamins,retinoids, and retinols (e.g., vitamin B₃, vitamin A, etc.); pigments;fragrances; sunscreens and sunblocks; anti-oxidants and radicalscavengers; organic hydroxy acids; exfoliants; skin conditioners;moisturizers; ceramides, pseudoceramides, phospholipids, sphingolipids,cholesterol, hyaluronic acid and its derivatives, collagen synthesispromoters, glucosamine, pharmaceutically acceptable penetrating agents(e.g., n-decylmethyl sulfoxide, lecithin organogels, tyrosine, lysine,etc.); preservatives; antimicrobial agents; amino acids such as proline,pyrrolidone carboxylic acid, its derivatives and salts, saccharideisomerate, panthenol, buffers together with a base such astriethanolamine or sodium hydroxide; waxes, such as beeswax, ozokeritewax, paraffin wax; plant extracts, such as Aloe Vera, cornflower, witchhazel, elderflower, or cucumber and combinations thereof. Other suitableadditives and/or adjuncts are described in U.S. Pat. No. 6,184,247, theentire contents of which are incorporated herein by reference.

Generally, the nonpolar GSE composition is topically applied at least ona daily basis for a period of time sufficient to bring about the desiredlevel of increased tan of skin or decreased gray appearance of hair.Topical application of the nonpolar GSE composition may continue for anysuitable period of time. More specifically, within a few days to withina few months of the initial application, a user may notice less grayhair and an increased tan appearance as well as an improvement in skinor hair texture and smoothness. It should be appreciated that thefrequency with which the composition disclosed herein should be appliedwill vary depending on the desired level of tan appearance or the amountof gray hair to treat. In particular, the degree of cosmetic enhancementwill vary directly with the total amount of composition used.

It is intended that the foregoing detailed description be regarded asillustrative rather than limiting. The present invention is furtherillustrated by the following experimental investigations and examples,which should not be construed as limiting. The contents of allreferences, patents and published applications cited throughout thispatent are hereby incorporated by reference herein.

EXAMPLES Example 1 GSE Stimulates Tyrosinase Gene Expression inMelanocytes

According to the present invention, a nonpolar GSE may be used tostimulate tyrosinase synthesis and expression in melanocyte cell linesin culture. Tyrosinase is a key enzyme in the melanin synthesis pathway.Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)can be used to measure relative levels of tyrosinase mRNA. For adescription of this technique see, for example, Kawasaki et al.,“Diagnosis of chronic myeloid and acute lymphocytic leukemias bydetection of leukemia-specific mRNA sequences amplified in vitro.” Proc.Natl Acad Sci USA. 1988 Aug;85(15):5698-702 and Livak et al.,“Oligonucleotides with fluorescent dyes at opposite ends providequenched probe system useful for detecting PCR product and nucleic acidhybridization.” PCR Methods Appl. 1995 Jun;4(6):357-62, which areincorporated herein by reference.

More specifically, in the present example, a mammalian melanocyte-likecell line, G-361, was treated with GSE at concentrations of 0.5, 5, and50 μg/ml in growth media and qRT-PCR levels of tyrosinase were comparedat 6 hours following treatment of G-361 with GSE and at 20 hoursfollowing treatment with GSE.

In particular, G-361 cells growing in their recommended growth media aretreated with GSE dissolved in a solution containing 50% DMSO:30%Ethanol:20% Water, which allows for a final solvent (GSE) concentrationnot exceeding 0.5%. Additionally, G-361 cells are maintained in a growthmedia stock solution containing 50% DMSO:30% Ethanol:20% water to whichno GSE was added, which serves as a negative control.

At six hours and twenty hours after treatment with GSE was initiated,G-361 cells from the GSE test samples and the negative control wereharvested to extract total mRNA to be used as template for thequantitative RT-PCR reaction. Fold induction was calculated by comparingthe level of expression of tyrosinase gene (TYR) and 18S rRNA in treatedversus untreated G-361 cells.

As shown in the FIG. 1, the regulation of the tyrosinase gene mediatedby GSE in G-361 melanocyte-like cells is dose dependent. The stimulatoryeffect became most significant at a concentration of about 50 μg/mlafter 20 hours of exposure. Generally, results showing over 2-foldregulation are significant as they represent a greater than 50% changein expression. Significantly, lower concentrations of GSE, for example0.5 .mu.g/ml, exhibited a small inhibitory effect on tyrosinase geneexpression at the 20 hour point.

Example 2 Fractionation of a GSE

GSE (e.g., Gravinol-S) was subjected to bio-directed fractionation. Inparticular, the GSE was subjected to a total extraction, a supercriticalfluid extraction, and a solvent sequential fractionation extraction,each of which are described more fully below.

More specifically, a total extraction procedure was performed using eachof ethanol; ethanol and water; chloroform; and acetone as a solvent witha powdered grape seed as follows:

a) Ethanol: Powdered grape seeds (2.06 g) were extracted in ethanol (20mL) under sonication at room temperature for 30 minutes. The sample wasfiltered under vacuum and supernatant was dried under nitrogen to afford442 mg of extract/fraction.

b) Ethanol/Water: Powdered grape seeds (2.01 g) were extracted inethanol/water, 1:1 v/v (20 mL) under sonication at room temperature for30 minutes. The sample was filtered under vacuum and supernatant wasdried under nitrogen to afford 450 mg of extract/fraction.

c) Chloroform: Powdered grape seeds (2.05 g) were extracted inchloroform (20 mL) under sonication at room temperature for 30 minutes.The sample was filtered under vacuum and supernatant was dried undernitrogen to afford 211 mg of extract/fraction.

d) Acetone: Powdered grape seeds (2.04 g) were extracted in acetone (20mL) under sonication at room temperature for 30 minutes. The sample wasfiltered under vacuum and supernatant was dried under nitrogen to afford195 mg of extract/fraction.

In addition, a GSE was obtained using a supercritical fluid extractiontechnique. In particular, supercritical fluid extraction was carried outusing carbon dioxide as the extracting solvent under supercriticalconditions. The extractions conditions were: extractor temperature 40°C., pressure 220 bars, CO₂ flow 20 g/minute, collector temperature 40°C., extraction time 30 minutes. Powdered grape seed sample (20g)afforded 1.79 g of a greenish yellow oil.

A sequential fractionation technique also was used to obtain a GSE. Inparticular, powdered grape seeds (2.04 g) were extracted in hexane (20mL) under sonication at room temperature for 30 minutes. The sample wasfiltered under vacuum and supernatant was dried under nitrogen to afford168 mg of hexane fraction. The ability of this hexane fraction to inducemelanin synthesis and tyrosinase expression was measured as described inExamples 4-6 below.

A portion of the hexane fraction was air dried and further subjected toextraction in ethyl acetate (20 mL) under sonication at room temperaturefor 30 minutes. The sample was filtered under vacuum and supernatant wasdried under nitrogen to afford 20 mg of ethyl acetate fraction. Theability of the ethyl acetate fraction to induce melanin synthesis andtyrosinase expression was measured as described in Examples 4-6 below.

A portion of the ethyl acetate fraction was air dried and furthersubjected to extraction in ethanol (20 mL) under sonication at roomtemperature for 30 minutes. The sample was filtered under vacuum andsupernatant was dried under nitrogen to afford 342 mg of ethanolfraction. The ability of the ethanol fraction to induce melaninsynthesis and tyrosinase expression was measured as described inExamples 4-6 below.

A portion of the ethanol residue was air dried and further subjected toextraction in ethanol/water 1:1 v/v (20 mL) under sonication at roomtemperature for 30 minutes. The sample was filtered under vacuum andsupernatant was dried under nitrogen to afford 184 mg of anethanol/water fraction. The ability of the ethanol/water fraction toinduce melanin synthesis and tyrosinase expression was measured asdescribed in Examples 4-6 below.

The residue was discarded after the above step.

Example 3 Induction of Melanin Synthesis by GSE Bio-DirectedFractionation Fractions

Different GSEs were obtained using bio-directed fractionation, asdescribed above in Example 2. In particular, the bio-directed fractionresulted in the following GSEs: an ethanol extract obtained by the abovedescribed total extraction procedure (“Ethanol-Total”) ; anethanol/water extract obtained by the total extraction procedure(“Ethanol/Water-Total”); a hexane extract obtained by the sequentialfractionation technique described above (“Hexane-Sequential”); an ethylacetate extract obtained by sequential fractionation (“EthylAcetate-Sequential”); an ethanol extract obtained by sequentialfractionation (“Ethanol-Sequential”); a chloroform extract obtained bythe total extraction technique; an acetone extract obtained by the totalextraction technique; an extract obtained from a supercritical fluidextraction; and a grape seed oil purchased from a grocery store. Theability of each of these extracts to induce melanin synthesis wastested.

In particular, G361 human melanoma cells were plated at 1×10⁵ /well in a12 well plate. The cells were treated with the samples identified belowin Table 3 at 50 μg/ml at 24 and 72 hours after plating. At 96 hoursafter plating, 1 M NaOH was added to the wells to extract cellularproteins. Extracts were boiled for 10 minutes. An aliquot of the extractwas assayed for protein content using a Coomassie blue protein assay.The OD₄₅₀ of the extracts was determined. The OD₄₅₀ was normalized tothe protein content of each sample and compared to that on untreatedcontrol cells. The results are reported below in Table 3:

TABLE 3 Melanin Induction Results % Control Melanin (Results normalizedto % Control Melanin viability and compared (Results compared to Sampleto melanin level in melanin level in untreated Description untreatedcontrol cells) control cells only) Ethanol-Total 103.2 95.7Ethanol/Water 120.8 72.9 Total Hexane 125.9 118.6 Sequential EthylAcetate 128.2 110.4 Sequential Ethanol 122.1 86.9 SequentialEthanol/Water 116.5 114.2 Sequential Supercritical 119.3 115.9 FluidExtraction Chloroform 123.8 114.9 Acetone 129.3 119.5 Grape Seed 106.295.5 Oil

As shown in Table 3, the non-polar fractions (hexane, ethyl acetate, andsupercritical fluid extraction) were the most potent at inducing melaninsynthesis.

Example 4 Induction of Tyrosinase Gene Expression by GSE

As explained above in Example 3, GSEs induce melanin synthesis inmelanocyte cell lines in culture. Therefore, the ability of each of theabove-identified GSEs to induce expression of tyrosinase, a key enzymein melanin synthesis, also was measured. In particular, relative levelsof tyrosinase mRNA were determined using quantitative reversetranscriptase-polymerase chain reaction (qRT-PCR). Tyrosinase mRNAlevels were measured in vitro and normalized to a housekeeping gene (18S rRNA).

In particular, G361 human melanoma cells were treated at a dosage of 50μg/ml with each of the following: an ethanol extract obtained by theabove described total extraction procedure (“Ethanol-Total”); anethanol/water extract obtained by the total extraction procedure(“Ethanol /Water-Total”); a hexane extract obtained by the sequentialfractionation technique described above (“Hexane-Sequential”); an ethylacetate extract obtained by sequential fractionation (“EthylAcetate-Sequential”); an ethanol extract obtained by sequentialfractionation (“Ethanol-Sequential”); an ethanol/water extract obtainedby sequential fractionation (“Ethanol/Water-Sequential”); a chloroformextract obtained by the total extraction technique; an acetone extractobtained by the total extraction technique; and a Gravinol-Snon-fractionated control extract.

More specifically, the G-361 cells were treated with the various GSEs ata dosage concentration of 50 μg/ml in a stock solution growth media. Thestock solution growth media was prepared in 50% DMSO:30% Ethanol:20%water to allow a final solvent concentration not exceeding 0.5% intreatment. The untreated control cells were fed a stock solution growthmedia of 50% DMSO:30% Ethanol: 20% water and no GSE was added.

Twenty hours after the beginning of treatment, the cells were harvestedto extract mRNA to be used as a template for subsequent quantitativeRT-PCR reaction. Fold induction was calculated by comparing the level ofexpression of tyrosinase and 18S rRNA in treated versus untreated G-361cells. The results are reported below in Table 4 and are shown in FIG.2.

TABLE 4 Tyrosinase Gene Induction Results Fold Change Relative to GSE:Untreated Control Ethanol - Total (mostly polar) −13.9 Ethanol/Water -Total (mostly polar) 20.7 Hexane - Sequential (nonpolar) 26.9 EthylAcetate - Sequential (mostly −19.8 nonpolar but can have a trace polar)Ethanol - Sequential (polar) 14.7 Ethanol/Water - Sequential (polar) 1.0Chloroform (mostly nonpolar but can −3.6 have a trace polar) Acetone(mostly nonpolar but can −3.3 have a trace polar) Gravinol-Snon-fractionated control 12.5 (mostly polar)

As shown above in Table 4 and in FIG. 2, tyrosinase gene inductionactivity was found in the ethanol-water, hexane, and ethanol fractionsof a GSE. In particular, the hexane fraction showed the best inductionof tyrosinase gene expression.

Similar results are shown in FIG. 3, which shows that a hexanesubfraction of a GSE had a significant impact on tyrosinase geneexpression. Specifically the procedure described above in this examplewas used to test sequential fractions of a GSE including an acetonefraction and methanol and hexane subfractions.

Example 7 Tyrosinase Inhibition Activity by GSE

GSEs are known to have an inhibitory activity on tyrosinase activity.Increased inhibition activity would be expected to reduce melanincontent in a melanocyte cell culture assay. Each of the following GSEswere assayed for tyrosinase inhibition activity: an ethanol extractobtained by the above described total extraction procedure(“Ethanol-Total”); an ethanol/water extract obtained by the totalextraction procedure (“Ethanol /Water-Total”); a hexane extract obtainedby the sequential fractionation technique described above(“Hexane-Sequential”); an ethyl acetate extract obtained by sequentialfractionation (“Ethyl Acetate-Sequential”); an ethanol extract obtainedby sequential fractionation (“Ethanol-Sequential”); an ethanol/waterextract obtained by sequential fractionation; a chloroform extractobtained by the total extraction technique; an acetone extract obtainedby the total extraction technique; and an extract obtained from asupercritical fluid extraction.

In particular, since tyrosinase catalyzes the conversion of L-DOPA todopachrome, in this example L-DOPA was used as a substrate and mushroomtyrosinase was used as a source of tyrosinase activity. Specifically,the conversion of colorless DOPA to dark orange dopachrome was monitoredat 475 nm. The optical density of dopachrome at 475 nm is directlyproportional to the intensity of orange color formation in solution(Beer-Lambert Law).

An extinction coefficient value of 3600 M⁻¹ cm⁻¹ was used to convertabsorbance units to activity. One unit of tyrosinase activity is definedas the amount required to produce one micromole of dopachrome per minuteat 25° C. and pH 6.5.

The results of this analysis are reported in FIG. 4. As shown in FIG. 4,in general, extracts of nonpolar solvents (e.g. hexane, ethyl acetate,and supercritical fluid extract) yielded little inhibition activitywhile extracts of polar solvents (e.g. acetone, ethanol, andethanol/water) exhibited inhibitory activity. The ethanol/waterfractions showed the greatest inhibition of tyrosinase activity.

Example 6 Stimulation of Melanin Synthesis in Gray or Graying Hair

A group of test subjects having gray hair is provided with a compositionof the present invention. Initially, a portion of the hair of eachparticipant in the group will be clipped close to the lower back of thescalp and natural melanin content for each subject will be analyzed andrecorded before any treatment with the composition of the presentinvention.

All test subjects will topically apply (to the portion of clipped hairat the back of the scalp) a standardized amount of the composition ofthe present invention. Such applications will occur daily for at leastthree months. Following at least three months of treatment, the portionof hair treated by topical application of the composition of the presentinvention will again be clipped and melanin content will be analyzed andrecorded. An increase in melanin content indicates that the compositionof the present invention is effective at stimulating melanin synthesis.

It should be understood that a wide range of changes and modificationscould be made to the compositions and methods of this invention. It istherefore intended that the foregoing description illustrates ratherthan limits this invention, and that it is the following claims,including all equivalents, which define this invention

1. A method of providing an increased skin tan appearance by increasingtyrosinase gene expression in skin cells comprising administering tosaid skin cells a composition comprising an effective amount of anon-polar grape seed extract, wherein the non-polar grape seed extractis obtained by: i) extracting powdered grape seeds with acetone toprovide an acetone extract; ii) extracting the acetone extract withhexane to produce a supernatant; and iii) drying the supernatant toprovide the non-polar grape seed extract, wherein the non-polar grapeseed extract increases tyrosinase gene expression.
 2. The method ofclaim 1, wherein the non-polar grape seed extract is present in anamount of about 0.01% to about 10% by weight of the composition.
 3. Themethod of claim 2, wherein the non-polar grape seed extract is presentin an amount of 1% to 9% by weight of the composition.
 4. The method ofclaim 2, wherein the non-polar grape seed extract is present in anamount of 4% to 6% by weight of the composition.
 5. The method of claim2, wherein the non-polar grape seed extract is present in an amount of2% to 3% by weight of the composition.
 6. The method of claim 1, whereinthe non-polar grape seed extract is administered in an amount rangingfrom approximately 30 μg/ml to approximately 150 μg/ml.
 7. The method ofclaim 6, wherein the non-polar grape seed extract is administered in anamount ranging from approximately 45 μg/ml to approximately 75 μg/ml. 8.The method of claim 6, wherein the non-polar grape seed extract isadministered in an amount of approximately 50 μg/ml.
 9. The method ofclaim 1, wherein the cell is a melanocyte.
 10. The method of claim 1,wherein the composition further comprises a cosmetically suitablevehicle.
 11. The method of claim 10, wherein the cosmetically suitablevehicle comprises water, ethanol, and butylene glycol.
 12. The method ofclaim 1, wherein the composition further comprises a penetrationenhancer that enhances penetration of the non-polar grape seed extractinto the skin cells.
 13. The method of claim 12, wherein the penetrationenhancer is a glycol, alcohol, or surfactant.